Total RNA was isolated from E13.5 cerebral cortex or from E13.5 cortical cells cultured in serum-free media for 20 h using Trizol reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol. The RNA was then treated with DNAse, primed with oligo (dT), and cDNA was synthesized using SuperScript II reverse transcriptase (Thermo Fisher Scientific). qPCR was carried out using a Bio-Rad CFX384 real-time PCR detection system, TaqMan probes (Applied Biosystems), and Taqman Fast Advanced Master Mix (Applied Biosystems). Transcripts for mouse Lpar1-6 and β-actin were detected with the following TaqMan probes, respectively: Mm01346925_m1, Mm00469562_m1, Mm00469694_m1, Mm01228533_m1, Mm02621109_s1, Mm00613058_s1, and Mm02619580_g1. Samples were measured in triplicate and the mouse β-actin probe was used for normalization to determine the relative expression of each gene by the 2−ΔCT method [44 (link)].
Cfx384 real time pcr detection system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The CFX384 Real-Time PCR Detection System is a high-throughput real-time PCR instrument designed for precise and accurate gene expression analysis. It features 384-well block capacity and advanced optical detection capabilities to enable efficient and reliable nucleic acid quantification.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (2 mentions)
Taqman probe, by Thermo Fisher Scientific (1 mentions)
Taqman fast advanced master mix, by Thermo Fisher Scientific (1 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Sybr green, by Thermo Fisher Scientific (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Quant studio 6 real time pcr instrument, by Thermo Fisher Scientific (1 mentions)
Cc 2512, by Lonza (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Cfx96, by Thermo Fisher Scientific (1 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Taqman universal master mix with no amperase uracil n glycosylase, by Thermo Fisher Scientific (1 mentions)
Taqman microrna assay, by Thermo Fisher Scientific (1 mentions)
4 protocols using cfx384 real time pcr detection system
1
Quantification of Lpar1-6 Expression in Developing Cortex
2
RNA Isolation and Real-Time PCR for Monocyte-Macrophage Differentiation
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RNA isolation was done as described for the gene expression microarray method. Briefly, RNA isolation was done using the miRNeasy Mini Kit (Qiagen), while random hexamers served as primers for first strand cDNA synthesis. The PCR was run on a CFX384 Real-Time PCR Detection System, and SYBRgreen (Applied Biosystems) was used to detect amplification of the PCR product. A list of primer sequences can be found in Table 1 . We analysed four housekeeping genes in the THP-1 monocytes and macrophages. Three out of four were unchanged by differentiation (Supplemental Figure S2a). From this, GAPDH was chosen for the analysis of primary monocytes differentiated to macrophages. Comparing different donors showed that GAPDH mRNA did not change upon differentiation (Supplemental Figure S2b). Therefore, the presented change in mRNA expression was calculated by the comparative copies per GAPDH.
3
Endothelial Gene Expression Analysis
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Total RNA was isolated from cell lines using the RNeasy Mini Kit (Qiagen) following manufacturer instructions and reverse transcribed to cDNA using the High-Capacity RNA-to-cDNA Kit (cat# 4387406; Thermo Fisher Scientific). Quantitative real-time PCR was performed using a BioRad CFX384 Real-Time PCR Detection System or Applied Biosystems QuantStudio6 Real-Time PCR instrument using TaqMan Gene Expression Assay reagents. The following Thermo Fisher Taqman gene expression assays were used to assess for endothelial gene expression in isolated CB-EPCs: Hs00990732_m1 (CD34), Hs00911700_m1 (KDR), Hs00174838_m1 (CD146), Hs01065279_m1 (PECAM1), Hs00923996_m1 (ENG), Hs00901465_m1 (CDH5), Hs00153304_m1 (CD44), Hs00243202_m1_(FSP1), and Hs99999905_m1 (glyceraldehyde 3-phosphate dehydrogenase). Expression of target genes was normalized to glyceraldehyde 3-phosphate dehydrogenase. The 2−ΔΔ Cycle threshold method was used to quantify the change in expression of the target genes relative to that in a standard control sample.30 (link) Human umbilical cord-derived mesenchymal stem cells (ATCC PCS-500–01) and normal human lung fibroblasts (Lonza, CC-2512) were used as nonendothelial cell sources for control.
4
Quantification of miR-126 Expression
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Reverse transcription of 10 ng of RNA was performed using TaqMan® microRNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA). Multiplex cDNA master mixes were prepared, whereby miR-126 primers were used in each reaction with the endogenous control RNU6B which were part of the TaqMan® microRNA Assays (Applied Biosystems, USA). RT-qPCR for miR-126 expression was performed using probes that are part of the TaqMan® microRNA Assays and 2× TaqMan® Universal Master Mix with no Amperase Uracil N-glycosylase (UNG) (Applied Biosystems, USA) on BioRad CFX96™ or CFX384™ Real-Time PCR Detection System (Hercules, CA, USA). The following steps were run: 10 min hold at 95 °C, 40 cycles of 15 s at 95 °C, and 60 s at 60 °C. miR-126 expression was normalized against the endogenous control RNU6B. Using the ΔΔCq, the relative expression of miR-126 was determined in the tumor samples compared to NAT and in the miR-126 mimic–transfected cells compared to the NC-transfected cells.
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